Rina Meidan

Impaired reproduction in heat-stressed cattle: basic and applied aspects

Summer heat stress (HS) is a major contributing factor in low fertility in lactating dairy cows in hot environments. Although modern cooling systems are used in dairy farms, fertility remains low. This review summarizes the ways in which the functioning of various parts of the reproductive system of cows exposed to HS is impaired. The dominance of the large follicle is suppressed during HS, and the steroidogenic capacity of theca and granulosa cells is compromised. Progesterone secretion by luteal cells is lowered during summer, and in cows subjected to chronic HS, this is also reflected in lower plasma progesterone concentration. HS has been reported to lower plasma concentration of LH and to increase that of FSH; the latter was associated with a drastic reduction in plasma concentration of inhibin. HS impairs oocyte quality and embryo development, and increases embryo mortality. High temperatures compromise endometrial function and alter its secretory activity, which may lead to termination of pregnancy. In addition to the immediate effects, delayed effects of HS have been detected as well. Among them, altered follicular dynamics, suppressed production of follicular steroids, and low quality of oocytes and developed embryos. These may explain the low fertility of cattle during the cool autumn months. Hormonal treatments improve low summer fertility to some extent but not sufficiently for it to equal winter fertility. A limiting factor is the inability of the high-yielding dairy cow to maintain normothermia. A hormonal manipulation protocol, which induces timed insemination, has been found to improve pregnancy rate and to reduce the number of days open during the summer.

Seasonal and acute heat stress effects on steroid production by dominant follicles in cows

The present study concerned the seasonal and acute effects of heat stress on steroid concentrations in follicular fluid and on steroid production by granulosa and theca interna cells, in bovine dominant follicles. Three groups of cows were studied: summer (n = 5), autumn (n = 5) and winter (n = 9) cows. During the winter season, another group of cows was acutely heat-stressed from days 3 through 5 of the estrous cycle (n = 5). On day 7 of the estrous cycle, follicular fluid from first-wave dominant follicles was aspirated, and dispersed granulosa and theca cells from each seasonal group were incubated for 18 h at normothermic (37.5 degrees C) or high (40.5 degrees C) temperatures. Cells were incubated in media only or in media containing testosterone (300 ng ml-1, for granulosa cells) or forskolin (4 micrograms ml-1, for theca cells). In follicular fluid the 17 beta-estradiol concentration was high (P < 0.05) in winter and low in autumn, and summer, the androstenedione concentration was high in summer (P < 0.05), low in autumn, and intermediate in winter. During the winter season, acute in vivo heat stress increased follicular fluid androstenedione and decreased estradiol to levels comparable with those prevailing in summer. Basal and forskolin-stimulated androstenedione production by theca cells was higher (P < 0.05) in the winter group than in the summer and autumn groups, and also higher than in the cows that were heat-stressed during winter, which suggests that theca cell function is susceptible to chronic (summer), short-term (winter) and delayed (autumn) heat stresses. In vitro incubation at high temperature (40.5 degrees C) reduced the high, forskolin-stimulated androstenedione production in winter (P < 0.05). Estradiol production by granulosa cells was high in winter and autumn, and low in summer (P < 0.05). Acute heat stress in winter did not alter estradiol production relative to winter controls, whereas a high incubation temperature (40.5 degrees C) reduced (P < 0.05) estradiol production only in the autumn, when the highest production rate was recorded. The results indicate a differential effect of heat stress on the functions of granulosa and theca cells. Both concurrent and delayed effects of heat stress on the steroidogenic capacity of ovarian follicles in cattle are presented.

Role of Tumor Necrosis Factor α and Its Type I Receptor in Luteal Regression: Induction of Programmed Cell Death in Bovine Corpus Luteum-Derived Endothelial Cells

Abstract The role of tumor necrosis factor α (TNFα) and its type I receptor (TNFRI) in structural luteolysis was investigated. A semiquatitative reverse-transcription polymerase chain reaction (RT-PCR) was used to characterize the pattern of TNFRI mRNA expression within the corpus luteum (CL) throughout the estrous cycle and its cellular distribution. Increase in TNFRI mRNA levels was recorded both in regressed luteal tissue and in CL of cows injected with prostaglandin F2α. All three major cell types composing the CL, steroidogenic (large and small) and endothelial cells expressed the TNFRI gene. A densitometric analysis of TNFRI mRNA expression revealed that resident endothelial cells had significantly higher levels of TNFRI mRNA than steroidogenic luteal cells. The physiological effects associated with TNFRI expression were investigated in the various luteal cell types. TNFα-induced programmed cell death (PCD) in dose- and time-dependent manners of cultured luteal endothelial cells (LECs) but not of in vitro luteinized steroidogenic cells. Several lines of evidence are provided to show that progesterone regulates luteal cell survival: 1) CL and LECs express progesterone receptor mRNA, 2) physiological levels of the steroid abolished TNFα-induced PCD of LECs, and 3) progesterone-producing cells are protected from PCD. In conclusion, this study suggests that TNFα-induced PCD during structural luteolysis is mediated by TNFRI, primarily affects endothelial cells, and that the decline in progesterone, preceding structural luteolysis, is a prerequisite for the initiation of apoptosis in endothelial cells.

Administration of Prostaglandin F2α During the Early Bovine Luteal Phase Does Not Alter the Expression of ET-1 and of Its Type A Receptor: A Possible Cause for Corpus Luteum Refractoriness

Abstract Luteal regression is initiated by prostaglandin F2α (PGF2α). In domestic species and primates, demise of the corpus luteum (CL) enables development of a new preovulatory follicle. However, during early stages of the cycle, which are characterized by massive neovascularization, the CL is refractory to PGF2α. Our previous studies showed that endothelin-1 (ET-1), which is produced by the endothelial cells lining these blood vessels, plays a crucial role during PGF2α-induced luteolysis. Therefore, in this study, we compared the effects of PGF2α administered at the early and mid luteal phases on ET-1 and its type A receptors (ETA-R) along with plasma ET-1 and progesterone concentrations, and the mRNA levels of PGF2α receptors (PGF2α-R) and steroidogenic genes. As expected, ET-1 and ETA-R mRNA levels were markedly induced in midcycle CL exposed to luteolytic dose of PGF2α analogue (Cloprostenol). In contrast, neither ET-1 mRNA nor its receptors were elevated when the same dose of PGF2α analogue was administered on Day 4 of the cycle. In accordance with ET-1 expression within the CL, plasma ET-1 concentrations were significantly elevated 24 h after PGF2α injection only on Day 10 of the cycle. The steroidogenic capacity of the CL (plasma progesterone as well as the mRNA levels of steroidogenic acute regulatory protein and cytochrome P450scc) was only affected when PGF2α was administered during midcycle. Nevertheless, PGF2α elicited certain responses in the early CL: progesterone and oxytocin secretion were elevated, and PGF2α-R was transiently affected. Such effects probably result from PGF2α acting on luteal steroidogenic cells. These findings may suggest, however, that the cell type mediating the luteolytic actions of PGF2α, possibly the endothelium, could yet be nonresponsive during the early luteal phase.

Deciphering the luteal transcriptome: potential mechanisms mediating stage-specific luteolytic response of the corpus luteum to prostaglandin F2α

The objective of this study was to identify prostaglandin F(2α) (PG)-induced changes in the transcriptome of bovine corpora lutea (CL) that are specific to mature, PG-responsive (day 11) CL vs. developing (day 4) CL, which do not undergo luteolysis in response to PG administration. CL were collected at 0, 4, and 24 h after PG injection on days 4 and 11 of the estrous cycle (n = 5 per day and time point), and microarray analysis was performed with GeneChip Bovine Genome Arrays. Data normalization was performed with affy package and significance testing with maanova from Bioconductor. Significance (relative to 0 h time point) was declared at fold change >2.0 or <0.5 and false discovery rate of <5%. At 4 and 24 h after PG, 221 (day 4) and 661 (day 11) and 248 (day 4) and 1,421 (day 11) regulated genes, respectively, were identified. The accentuated gene expression response in day 11 CL was accompanied by specific enrichment of PG-regulated genes in distinctive gene ontology categories (immune related and other), particularly at 24 h after injection. Specificity in putative transcription factor binding sites was observed among PG-regulated genes on day 11 vs. day 4, including a potential association of ETS transcription factors with acute PG-induced gene expression specific to day 11 CL. Temporal and PG-induced regulation of abundance of mRNA for ETS transcription factor family members linked to the stage-specific response to PG was not observed. Increased abundance of protein and/or mRNA for six PG-regulated putative ETS-responsive genes was noted in day 11 but not day 4 CL. Results reveal insight into stage-specific gene expression in bovine CL in response to PG and potential transcriptional mediators of luteolysis.

Regulation of Angiogenesis-Related Prostaglandin F2alpha-Induced Genes in the Bovine Corpus Luteum

ABSTRACT We recently compared prostaglandin F2alpha (PG)-induced global gene expression profiles in PG-refractory, bovine corpus luteum (CL) collected on Day 4 of the estrous cycle, versus PG-responsive, Day 11 CL. Transcriptome analyses led us to study the regulation of angiogenesis-related genes by PG and their functions in luteal endothelial cells (ECs). We found that PG regulated angiogenesis-modulating factors in a luteal stage-dependent way. A robust increase in FGF2 expression (mRNA and protein) occurred in the PG-refractory Day 4 CL promoting CL survival and function. Inhibitors of FGF2 action, thrombospondin 1 and 2, their receptor (CD36), and PTX3 were upregulated by PG specifically in Day 11 CL undergoing luteolysis. VEGF mRNA decreased 4 h post-PG in both Day 4 and Day 11 CL. The resulting destabilization of blood vessels in Day 11 CL is expected to weaken the gland and reduce its hormonal output. These genes were expressed in dispersed luteal ECs and steroidogenic cells; however, thrombospondin 1 and FGF2 were more abundant in luteal ECs. Expression of such genes and their ability to modulate FGF2 actions were investigated. Similar to its in vivo effect, PG, in vitro, stimulated the expression of thrombospondins and PTX3 genes in several luteal cell models. Importantly, these factors influenced the angiogenic properties of luteal ECs. FGF2 dose-dependently enhanced cell migration and proliferation, whereas thrombospondin 1 and PTX3 inhibited FGF2 actions in luteal ECs. Collectively, the data presented here suggest that, by tilting the balance between pro- and antiangiogenic factors, PG can potentially control the ability of the CL to resist or advance toward luteolysis.

Induction of Endothelin-2 Expression by Luteinizing Hormone and Hypoxia: Possible Role in Bovine Corpus Luteum Formation

The pattern and regulation of endothlin-2 (EDN2) expression and its putative roles in bovine ovaries were investigated. EDN2 mRNA was determined in corpus luteum (CL) and during folliculoluteal transition induced by GnRH in vivo. EDN2 was elevated only in the early CL and was not present in older CL. In the young CL, EDN2 mRNA was identified mainly in luteal cells but not endothelial cells that expressed the EDN1 gene. Similarly, in preovulatory follicles, EDN2 was expressed in the granulosa cells (GCs) and not in the vascular theca interna. LH and hypoxia are two major stimulants of CL formation. Therefore, GCs were cultured with bovine LH, under hypoxic conditions. GCs incubated with bovine LH resulted in increased EDN2 mRNA 42 h later. CoCl2, a hypoxia-mimicking agent, elevated EDN2 in GCs in a dose-dependent manner. Incubation of the human GC line (Simian virus 40 large T antigen) under low oxygen tension (1%) augmented EDN2 6 and 24 h later. In these two cell types, along with EDN2, hypoxia augmented VEGF. EDN2 induced in GCs changes that characterize the developing CL: cell proliferation as well as up-regulation of vascular endothelial growth factor and cyclooxygenase-2 (mRNA and protein levels). Human chorionic gonadotropin also up-regulated these two genes. Small interfering RNA targeting EDN-converting enzyme-1 effectively reduced its mRNA levels. This treatment, expected to lower the mature EDN2 peptide production, inhibited VEGF mRNA levels and GC numbers. Together these data suggest that elevated EDN2 in the early bovine CL, triggered by LH surge and hypoxia, may facilitate CL formation by promoting angiogenesis, cell proliferation, and differentiation.

Seasonal differences in progesterone production by luteinized bovine thecal and granulosa cells

This study examined seasonal differences in progesterone (P4) production by granulosa cells (GC) and thecal cells (TC) that were luteinized in vitro during the winter or the summer; it also compared plasma P4 concentrations of lactating dairy cows in the two seasons. First-wave dominant follicles obtained from Holstein cows were dissected on day 6 of the cycle, GC and TC were separated, enzymatically dispersed, and cultured for 9 days in media containing 1% fetal calf serum, forskolin (10 micromol/mL) and insulin (2 microg/mL), to induce cell luteinization. All experimental procedures were identical and characteristics of the follicles were similar in the two seasons. During 9 days of culture, P4 production by luteinized GC was higher in winter than in summer, but the difference only tended to be significant. In contrast, luteinized TC produced three times as much P4 in winter as in summer (324 versus 100 ng/10(5)cells). In the in vivo experiment, P4 concentrations in plasma collected during entire estrous cycles in winter and summer were compared. The cows were, on average, at 70 days postpartum and yielded similar amounts of milk. Concentrations of progesterone in plasma were significantly higher in winter than in summer; during the mid-luteal phase the difference between the two seasons was 1.5 ng/mL. These results indicate that chronic effects of heat-stress are possibly carried over from an impaired follicle to an impaired corpus luteum (CL), and that luteinized TC are more susceptible to heat-stress than luteinized GC.

The ovarian endothelin network: an evolving story

The endothelin (ET) system consists of three ET isopeptides, several converting enzyme isoforms and two G-protein-coupled receptors, ETA and ETB, which are linked to multiple signaling pathways. Less than 20 years after the initial detection of ET-1 in granulosa cells, the ovarian ET network continues to expand with the discovery of new members and functions. ETs influence a broad range of essential reproductive processes, such as ovulation, steroidogenesis and luteolysis. Therefore, a more comprehensive understanding of the ovarian ET network might provide new strategies for controlling reproduction. This review presents up-to-date findings on the ET network in the ovary.

Subclinical, chronic intramammary infection lowers steroid concentrations and gene expression in bovine preovulatory follicles

Chronic, subclinical intramammary infection depresses fertility. We previously found that 30% of subclinical mastitic cows exhibit delayed ovulation, low circulating estradiol levels, and delayed luteinizing hormone surge. We examined the function of preovulatory follicles of cows experiencing subclinical mastitis or a past event of acute clinical mastitis. Cows were diagnosed for mastitis by somatic cell count and bacteriological examination. All clinical infections were caused by Escherichia coli, and most subclinical infections were caused by Streptococcus dysgalactiae and coagulase-negative staphylococci. On day 6 of the cycle, cows received PGF2α; 42 h later, follicular fluids and granulosa cells or theca cells were aspirated from preovulatory follicles in vivo or following slaughter, respectively. Overall, follicular estradiol and androstenedione concentrations in the subclinical group (n = 28) were 40% lower (P < 0.05) than those in uninfected cows (n = 24) and lower than in past clinical mastitic cows (n = 9). Distribution analysis revealed a clear divergence among subclinical cows: one-third (9/28) exhibited low follicular estradiol; the other two-thirds had normal levels similar to all uninfected (P < 0.01) and most clinical cows (P < 0.08) that had normal follicular estradiol levels. Subclinical normal-estradiol cows had twofold higher (P < 0.05) circulating estradiol concentrations and sevenfold and fourfold higher (P < 0.05) follicular androstenedione levels and estradiol-to-progesterone ratio, respectively, than subclinical low-estradiol cows. Follicular progesterone level was not affected. Reduced expression (P < 0.05) of LHCGR in theca and granulosa cells, CYP11A1 (mRNA and protein) and CYP17A1 in theca cells, and CYP19A1 in granulosa cells may have contributed to the lower follicular steroid production in the subclinical low-estradiol subgroup. StAR and HSD3B1 in theca cells and FSHR in granulosa cells were not affected. Mastitis did not alter follicular growth dynamics, and no carryover effect of past clinical mastitis on follicular function was detected. These data indicate that a considerable proportion (one-third) of subclinical mastitic cows have abnormal follicular steroidogenesis, which can explain the reproductive failure associated with this disease.